The present invention relates to a multi-point light-delivering device, comprising a waveguide carrying light along a longitudinal axis and including multiple optical windows, through Which the carried light is out-coupled from the waveguide.
Such a device may be used as an optogenetic tool to be exploited both for in-vitro experiments with neuronal tissues and for in-vivo experiments or medical applications, in such experiments, specific neurons are targeted to express light-sensitive proteins or are exposed to light-sensitive compounds. Examples of light-sensitive proteins include proteins that alter the electrical and biochemical state of the neuron or that activate or repress specific enzymes. Light-sensitive compounds include small molecules that when exposed to light release an active compound such as a neurotransmitter, second messenger, or neuromodulator. Thus the electrical, biochemical, and signalling state of a neuron can be regulated by optical stimulation, typically in the visible spectral range or in the near infrared. The main advantage of optical stimulation compared to classical electrical or pharmacological stimulation is the possibility to selectively activate or inactivate (one or more) genetically defined set(s) of neurons with high temporal and spatial precision. This can be achieved by genetic approaches that can provide specific neurons with light-sensitive proteins while leaving neighbouring cells insensitive. In contrast, electrical or pharmacological stimulation generally affects all cells located near the electrode tip, with no cellular precision.
Optical stimulation in brain tissue is often performed simultaneously with electrical recording of the triggered neuronal activity. In in-vivo experiments, common optogenetic tools are optical fibers used to shine visible light inside the brain, combined with single- or multi-electrode recording systems (single microwires, tetrodes, multielectrode arrays fabricated on silicon shafts, etc.) for the electrical readout. These tools are managed separately, and neural responses can be monitored near and far from the optical stimulation region. In recent years, fully integrated devices combining optical modulation and electrical recording in a single implantable tool have been developed, thus improving compactness and reducing surgical intervention steps.
To understand the behaviour of complex neural circuits and to increase the amount of data collected in single experiments, multiple-channel recording is crucial. However, in standard devices light is delivered into the brain by means of a single optical fiber able to deliver light only to a single, fixed area of the brain. The high number of recording channels is thus not accompanied by comparable amount of light delivery points, creating a mismatch in which electrical activity can be detected with high spatial resolution, while the optical excitation has a poor spatial selectivity. The possibility to dynamically select the excited area in real time would boost the performances of currently available devices, allowing more flexible and powerful causal manipulation of neural circuits.
Recently, integrated single optical excitation/multiple electrical readout systems have been reported in Anikeeva et al. (Nature Neuroscience, vol. 15, pp. 163-170, 2012, doi: 10.10381/nn.2992) and Wang et al. (Journal of Neural Engineering, vol. 9, p. 016001, 2012, doi: 10.1088/1741-2560/9/016001). In Anikeeva et al., a single multimodal fiber is used to convey light, while four tetrodes are glued on its sides and extended 300 μm or more beyond the tip of the optical fiber to record electrical signal from illuminated brain regions. The system is compact and lightweight, suitable for chronic implantation on freely moving animals. However, this layout limits localization of the recording sites to a small brain region near the tip of the fiber, with the effective distance determined by the light intensity. The absorption and scattering of light in brain tissue leads to a decrease in light intensity as a function of distance from the fiber tip: electrode tips closer than 200-300 μm from the fiber tip will suffer from high Photoelectric noise, while sensors farther than 1000 μm will generally be outside of the range of light illumination. The effective region will be therefore limited to few hundreds microns from the fiber tip.
The second approach proposes a tapered optical fiber positioned at the center of a two-dimensional microelectrode array consisting of 30 microfabricated silicon tips for electrical recording. The optical fiber is tapered only for the purpose of preventing tissue damages. The fiber can also be covered by a metallic layer to provide an additional electrical recording site (see also Zhang et al, Journal of Neural Engineering, Vol. 6, p. 055007, 2009, doi: 10.1088/1741-2560/6/5/055007). Inter-electrode distance and minimum electrode-fiber distance is determined by the microfabrication (in the proposed device, it is 400 μm). Light intensity is adjusted to excite neurons from the tip of the fiber to the closer electrodes of the array. The excited brain volume can again he expanded by increasing the emitted optical power, but increasing light intensity to reach distant recording sites will lead to increased electrical artifacts on the closer sites.
Commercially available optrodes (NeuroNexus) also combine a linear array of recording sites fabricated on a single silicon shaft with a hare optical fiber collinearly mounted on top of the array (see also Royer at al., European Journal of Neuroscience, Vol. 31, pp. 2279-2291, 2010, doi: 10.1111/j.1460-9568.2010.07250.x). Again, the electrodes/fiber tip distance is determined by the light intensity: closer distances require lower optical power to avoid photoelectric noise; therefore reduced brain volumes are excited. Typically, intermediate fiber distances of 200 μm from the closest pad are provided as a commercial standard.
It is therefore evident that optrodes based on a single light-emitting point source have significant limitations for the integration of multiple-site recording systems. Multi-point light delivery has been proposed. by Zorzos et al. (Optics Letters, Vol. 15, pp. 4133-4135, 2010; Optics Letters, Vol. 37, pp. 4841-4843, 2012) and Stark et al. (Journal of Neurophysiology, vol. 108, pp. 349-363, 2012). The approaches of Zorzos et al. comprise a parallel array of optical waveguides having a 45° terminal cut covered by aluminium, so that 90° light emission, perpendicular to the probe axis, is obtained. Each waveguide can be separately coupled to different light sources or to the same laser source shared by all the waveguides by moans of micro-mirror devices, therefore obtaining separate optical stimulation points in two- and three-dimensional environments. Although electrical recording is not described in these publications, integration with silicon shafts and multiple recording sites is suggested. Stark et al. also propose the use of multiple diode-fiber assemblies, where each single-core optical fiber is glued to a different silicon shaft with single or multiple recording sites. In this case, each fiber is independently excited and multiple wavelengths and light powers can be used. Both strategies allow an improved distribution of light intensity in the investigated brain volume, but this is obtained by recurring to multiple light sources and complicated and cumbersome coupling strategies.
WO 2011/057137 discloses a waveguide neural interface device able to target different brain regions. It is based on the combination of sonic of the above described works and it covers a wide area of possible device configurations. In particular, light directing elements are provided on waveguides and/or on optical fibers in order to redirect light away for the longitudinal axis of the waveguide. These elements allow the illumination of specific zones of the tissue surrounding the device and, as stated by the inventors, can be “one or more of several variations, including one or more features that refract, reflect, focus, and/or scatters light, and/or perform any suitable manipulation of light”. That is, light is redirected and/or manipulated by means of light-directing elements realized on a waveguide, while the purpose of the waveguide is just to carry light to the reflecting elements. According to WO 2011/057137, the waveguide could he tapered to reduce tissue damages.
The device configuration disclosed by WO 2011/057137 may he somewhat complex and cumbersome, particularly when a great number of light-directing elements must be provided with the waveguide.
One object of the invention is therefore to provide a multi-point light-delivering device that overcomes the drawbacks of existing devices,